DEVELOPMENTAL DYNAMICS 239:3275–3284, 2010
a
TECHNIQUES
Simple and Efficient Transgenesis with I-SceI Meganuclease in the Newt, Cynops pyrrhogaster
Developmental Dynamics
Martin Miguel Casco-Robles, Shouta Yamada, Tomoya Miura, and Chikafumi Chiba*
Newts have been recognized as an ideal model for body-parts regeneration after traumatic injury since the 18th century. However, molecular mechanisms underlying regeneration remain a mystery because of technical limitations. In the current study, to break this obstacle, we established a simple and efficient transgenic protocol for the newt Cynops pyrrhogaster by adapting an I-SceI microinjection technique, as well as a two-aquarium-tank (TAT) system that allows us to constantly obtain fertilized eggs in the laboratory for transgenesis. Following our protocol, ~20% of injected embryos would exhibit non-mosaic widespread transgene expression and survive beyond metamorphosis. This anticipated success rate is about 10 times higher than that obtained by previous protocols, reaching a practical level. Therefore, our transgenic protocol in conjunction with the TAT-system could provide a key technique to open the way to uncover the long mystery underlying body-parts regeneration of newts. Developmental Dynamics 239:3275–3284, 2010. V 2010 Wiley-Liss, Inc. C
Key words: newt; transgenesis; I-SceI; meganuclease; EGFP Accepted 24 September 2010
INTRODUCTION Newts are a subset of the family Salamandridae of urodele amphibians (Weisrock et al., 2006) and have been recognized as a useful model animal since the 18th century, contributing to the progress of various research fields in biology and medicine (Colucci, 1891; Wolff, 1895; Spemann and Mangold, 1924; Taguchi et al., 1989; Kurahashi, 1990; Okada, 1991; Kikuyama et al., 1995; Mitashov, 1996). Especially for studies on tissue repair/ regeneration after a traumatic injury, newts are indispensable animals because of their unique and outstanding ability: they can regenerate, even in adulthood, missing body-parts such as eye tissues including the retina
and lens, and a part of the brain and heart as well as jaws, limbs, and tail, through a mechanism of transdifferentiation during which terminally differentiated somatic cells convert into multipotent stem-like cells (Goss, 1969; Brockes and Kumar, 2002; Tsonis and Del Rio-Tsonis, 2004; Chiba and Mitashov, 2007). Unfortunately, however, the molecular mechanisms underlying such intriguing phenomena remain to be elucidated, since, in this animal, the development of tools and techniques to manipulate gene functions lag behind that for other model organisms. So far, to analyze gene functions in vivo, researchers have tried to deliver molecular tools such as plasmid vectors
(for gain of function) or antisense oligonucleotides (for loss of function) into cells in living animals by in situ transfection using chemical reagents (Madhavan et al., 2006), electroporation (Kumar et al., 2007), or particle bombardment (Pecorino et al., 1996), or by implantation of cells that have been transfected in culture (Hayashi et al., 2001; Chiba et al., 2004; Grogg et al., 2005). However, these methods have some limitations such that it is difficult to deliver molecular tools to a target cell population uniformly. Moreover, the effects of such tools are not stable, sometimes causing inconsistent consequences. On the other hand, transgenesis can be a hopeful technique to overcome such weakness and allow us to
Additional Supporting Information may be found in the online version of this article. Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan Grant sponsor: Japan Society for the Promotion of Science (JSPS); Grant numbers: 20650060, 21300150. *Correspondence to: Chikafumi Chiba, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki 305-8572 Japan. E-mail:
[email protected] DOI 10.1002/dvdy.22463 Published online 27 October 2010 in Wiley Online Library (wileyonlinelibrary.com).
C 2010 Wiley-Liss, Inc. V
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regulate gene functions at a given time and place in vivo. To our knowledge, transgenesis of newts has been tried with a Japanese common newt (fire bellied newt; Cynops pyrrhogaster; see Fig. 1). Makita et al. (1995) reported for the first time transgenic newts produced by microinjection of DNA constructs into fertilized eggs. However, unfortunately, those larvae showed highly mosaic expression patterns of exogenous genes. To circumvent this problem, Ueda et al. (2005) applied a procedure established by Kroll and Amaya (1996) for Xenopus, i.e., transplantation of sperm nuclei carrying transgenes into unfertilized eggs, with some critical modifications, and thereby succeeded in producing transgenic newts exhibiting widespread non-mosaic expression of exogenous genes in embryos, swimming larvae, and juveniles after metamorphosis. However, in the meantime, efficiency of transgenesis after microinjection of DNA constructs into fertilized eggs was substantially improved in other model animals by the introduction of the ISceI meganuclease technique established in a fish, medaka Oryzias latipes (Thermes et al., 2002). In this approach, I-SceI meganuclease, which is an intron-encoded homing endonuclease isolated from the yeast Saccharomyces cerevisiae (Jacquier and Dujon, 1985), is co-injected with DNA constructs carrying a transgene cassette flanked by 18-bp I-SceI recognition sites. After microinjection, this enzyme facilitates integration of foreign genes into the host genome, allowing the generation of stable transgenic lines. Thus far, this approach has been applied successfully to several fish species [medaka (Thermes et al., 2002); stickleback Gasterosteus aculeatus (Hosemann et al., 2004); zebrafish Danio rerio (Grabher et al., 2004)], amphibians [axolotl Ambystoma mexicanum (Sobkow et al., 2006); Xenopus laevis (Pan et al., 2006); Xenopus tropicalis (Ogino et al., 2006)], and ascidians [Ciona savignyi (Deschet et al., 2003)]. If this approach were to also work in the newt, it could be an alternative way of newt transgenesis with higher efficiency and convenience. Therefore, in the current study, we attempted to apply the I-SceI meganuclease technique to the C. pyrrhogaster newt.
Fig. 1. Fire bellied newt Cynops pyrrhogaster in the mating season. A: Female. B: Male. The male characteristically has a purple tail with black spots.
RESULTS AND DISCUSSION Two-Aquarium-Tank System Is Useful to Obtain Fertilized Eggs in the Laboratory For successful transgenesis, it is critical to obtain fertilized eggs efficiently. However, in the C. pyrrhogaster newt, the quality and quantity of eggs/ sperms are highly dependent on animals that are collected from the field (also pointed out in Ueda et al., 2005). Moreover, this species is seriously decreasing in number (press release in 2006, Ministry of Environment of Japan; http://www.env.go.jp/press/press. php?serial¼7849). Therefore, here we attempted to obtain fertilized eggs in the laboratory by setting up an aquarium system that allows the animals to grow and mate as in the fields. We prepared the aquarium tank (60-cm width; 30-cm depth; 45-cm height) with filtration/circulation and heater/thermostatic systems, and placed them beside glass windows away from direct sunlight in an animal stock room kept at 18 C (Fig. 2A). Small rocks were placed in the tank to allow animals to hide and rest and the water was filled up to 15 cm in
depth. The heater/thermostatic system was set at 14 C. At the beginning of the mating season (November 2009), adult newts captured during the last mating season (November 2008 and April 2009) were transferred into the tank and kept at 14– 18 C under natural light conditions through the windows. The number of females per tank was fixed at 30, and the male to female ratio was set at 1–2:3 because ratios of males lower than this range reduced males attractive for females, while higher ratios of males caused competition/disturbance between males, both leading to less successful mating. The filtration/circulation system was operated only to clean the tank (4–6 hr/day) so as to keep the water still for the majority of time. Under these conditions, animals displayed a characteristic courtship behavior followed by delivery of a spermatophore from male to female (Tsutsui, 1931; Kikuyama and Toyoda, 1999). From one month later (December 2009), every female was injected with a hormone gonadotropin every other day (see Experimental Procedures section). A few days after the first injection, females started to lay eggs on strips of plastic sheets. Females that laid
I-SceI TRANSGENIC PROTOCOL FOR NEWT 3277
weeks during which time we collected eggs from the other tank (Tank-2). Using this two-tank system, we could obtain eggs continuously for longer than 7 months (typically, 60–80 eggs/ round) (Fig. 2C). The fertilization ratio estimated by cleavage was higher than 70%. We used these fertilized eggs in the following experiments.
Optimal Microinjection Site Is Around the Animal Pole
Developmental Dynamics
Subsequently, as the groundwork for microinjection, we attempted to determine the best injection site by evaluating it with the survival rate until the swimming larvae (SL) stage (Fig. 3; for controls with no microinjection, see Supp. Tables S1 and S2, which are available online). Around the top (i.e., the animal pole) and side of the pigmented area of the fertilized egg (one-cell stage embryo) were tested because the intracellular space under this region contains the cytoplasm and pronucleus (Fig. 3A). A small amount (approximately 20 nl) of a tracer solution (0.01% phenol red in water) was injected through a glass micropipette whose tip was grinded to an angle of 35 s (Fig. 3B), and the injected embryos were reared at room temperature (22–24 C). When the solution was injected near the top of the pigmented area (Fig. 3C), 29% of embryos (n ¼ 48) survived until the SL-stage, whereas embryos (n ¼ 61) injected at the side of the pigmented area died before the neurula (N) stage, indicating that the microinjection site influences the survival of embryos (Fig. 3D). We also tested the non-pigmented region in the vegetal pole side, but injection was quite difficult because yolk plugged up the micropipette tip. Therefore, we decided to make microinjections near the top of the pigmented area. Fig. 2. Two-aquarium-tank system for egg collection. A: Aquarium-tank system. B: Schematic graphs showing the schedules of hormone gonadotropin injection (H) and collection of eggs using two-aquarium-tank system. The number of eggs collected in a tank declines in 2 weeks. Therefore, to obtain eggs constantly, two tanks were used alternately each 2 weeks. C: The number of eggs collected on the day of experiment (square) and the percentage of fertilized eggs in them. The averaged values calculated monthly are shown. Vertical bars indicate SEM. N, the number of rounds.
abnormal eggs and dead animals were replaced with healthy ones. The number of eggs collected from the same tank decreased gradually over 2 weeks. Therefore, we used two
tanks, alternating them every 2 weeks (Fig. 2B); after we collected eggs from one tank (Tank-1) for 2 weeks, we allowed females there to recover without hormone treatment for the next 2
Incubation of I-SceI Meganuclease and DNA Constructs Before Microinjection Is Critical for Non-Mosaic Dispersed Expression of Exogenous Genes in Embryos We tried to microinject a mixture of a reporter DNA construct pCAGGs-
3278 CASCO-ROBLES ET AL.
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Subsequently, we examined the influence of DNA-I-SceI concentration per embryo on the expression pattern. We injected 4 nl of the same solution (i.e., 0.4 ng DNA and 0.004 U I-SceI), that was pre-incubated at 37 C for 40 min, into one-cell embryos. Under this condition, B-stage survivors showing uniform expression patterns further increased to 55.4%, while those showing mosaic patterns or no green fluorescence decreased (Table 1). In conclusion, this condition was better than before, although the % survivors showing uniform expression patterns with strong green fluorescence seemed not to be affected.
Rearing Temperature After Microinjection Significantly Influences the Survival of Embryos Fig. 3. Optimization of the microinjection site. A: Schematic diagram showing the injection sites tested. Microinjection was tried around the top (a) and the side (b) of the pigmented hemisphere of a fertilized egg (one-cell embryo). B: The tip shape of a micropipette for injection. Inset is a magnified view of the tip. The scale on the ruler is 30 mm. C: A view of microinjection around the top of the egg. Scale bar ¼ 1 mm. D: Effects of the site of microinjection on the survival of embryos. A tracer solution (20 nl) was injected around either the top or the side of the one-cell embryos. When the solution was injected near the top, 29.4% of embryos (n¼48; N¼2) survived until the swimming larvae (SL) stage, whereas embryos (n¼61; N¼2) injected at the side died before the neurula (N) stage. Note that the embryos after microinjection were reared at room temperature (22–24 C). C, cleavage; B, blastula; N, neurula; TB, tail-bud; SL, swimming larvae.
EGFP (Sce) and I-SceI meganuclease into one-cell-stage embryos. This construct (5.9 kb) contains the pCAGGsEGFP cassette flanked by I-SceI recognition sites that express EGFP under the control of chicken b-actin promoter with CMV-IE, and has been applied successfully to axolotl transgenesis (Sobkow et al., 2006). We initially prepared a microinjection solution containing 0.1 mg/ml of the DNA construct, 1 unit (U)/ml of I-SceI enzyme, 0.01% phenol red, and 1 I-SceI buffer, and injected 20 nl solution (i.e., 2 ng DNA and 0.02 U I-SceI) into the one-cell embryos, and then reared them at room temperature (22–24 C). Under this condition, most embryos started expression of EGFP from the blastula (B) stage (Fig. 4A, B). We examined expression patterns at the B-stage (Table 1), and found that 11.4% of survivors expressed EGFP uniformly (Fig. 4A); however, 54.1% exhibited mosaic patterns (Fig.
4C). For expression categories, see the Data Analysis section in the Experimental Procedures section. To improve this result, we incubated the injection solution at 37 C before microinjection, since insufficient enzymatic reaction of I-SceI meganuclease to DNA constructs has been suggested to be a cause of such mosaic expression patterns (Ogino et al., 2006). Expectedly, when the solution was incubated for 40 min, Bstage survivors showing uniform expression patterns increased to 38.4% (more than three-fold) [strong expression: 13.8% (8.6-fold); moderate/weak expression: 24.6% (2.5fold)], while those showing mosaic patterns or no green fluorescence decreased (Table 1). We tested different incubation times (15, 30, 40, 60 min) on the expression pattern. Finally, a 40-min incubation yielded optimal results to obtain embryos with uniform expression patterns.
For practical transgenic protocols, high levels of survival rate of embryos after microinjection are required as are uniform expression patterns. Unfortunately, under the conditions we examined so far, most embryos died before the N-stage (Table 2). As a cause of death, we suspected mechanical damage itself in the microinjection because similar phenomena were observed even after microinjection of the tracer only (Fig. 3D). Therefore, to overcome this serious problem, we subsequently explored optimal rearing conditions that would allow the injured embryos to recover. Here, to evaluate effects of parameter changes on mechanical damage, we used onecell embryos that underwent only a poke with the tip of the micropipette (but not injection). We examined the influence of rearing temperature on the survival of embryos. For that, we reared the poked embryos at lower temperatures (18 or 14 C). Surprisingly, under these conditions, most embryos survived beyond the N-stage (Table 3). When the poked embryos were reared at 14 C, the survival rate at the SLstage dramatically improved to 81.8%; the value was higher than that (55.3%) at 18 C and close to the control value (88.5%) with no poke (Supp. Table S2). Subsequently, to examine the time for recovery, we changed the time during which the poked embryos
Developmental Dynamics
I-SceI TRANSGENIC PROTOCOL FOR NEWT 3279
Fig. 4. Transgenic newts generated by the I-SceI method with pCAGGs-EGFP(Sce) construct. A, D–G: Embryos showing an intense green fluorescence uniformly at blastula (A), neurula (D, neural plate stage; E, neural tube stage), tail-bud (F), and swimming larvae stage (G). B: Bright-light image of the same field in A. C: A blastula embryo showing a mosaic expression pattern (arrowhead). H: A magnified view of the head region of the larva in G. I–L: A juvenile newt after metamorphosis [anesthetized in 0.1% FA100 (Tanabe, Japan) for 8–10 min]. Dorsal (I, J) and ventral (K, L) views are shown. I, K: Fluorescence image; J, L: Bright-light image. For movies of the larva in G, H and the juvenile in I–L, see Supp. Movies S1 and S7, S8. Scale bars ¼ 1 mm (A–F), 3 mm (G), 1.2 mm (H), 1 cm (I–L).
were incubated at 14 C (Fig. 5). The survival rate at the SL-stage tended to increase by extending the incubation time (Jonckheere-Terpstra test: P ¼ 0.0004). The mean value was significantly increased with an incubation
time longer than 24 hr [24 h, 61.7 6 4.4, N ¼ 3, n ¼ 33; 1 (until stage 35; Supp. Fig. S1), 82.0 6 0.0, N ¼ 3, n ¼ 33] compared to that with 1-hr incubation (Kruskal-Wallis test followed by Fisher’s LSD test: 24 h, P ¼ 0.018;
1, P ¼ 0.001). These results suggest that the poked embryos recover from mechanical damage in temperatureand time-dependent manners. In conclusion, after microinjection rearing embryos at 14 C until stage 35 could
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TABLE 1. EGFP Expression Patterns at the Blastula Stagea EGFP expression patterns Injection DNA-I-SceI No. survivors Uniform volume incubation at blastula Strong Moderate
Mosaic
ND
(nl/egg)
time (min)
stage
(%)
(%)
(%)
20 20 4
0 40 40
61 65 83
1(1.6) 6(9.8) 9(13.8) 16(24.6) 10(12.0) 36(43.4)
Weak (%)
33(54.1) 21(34.3) 30(46.2) 10(15.4) 21(25.3) 16(19.3)
Injection solution contained the DNA construct (0.1 mg/ml) and I-SceI enzyme (1 U/ ml). Embryos after microinjection were reared at room temperature (22–24 C). Survival rate of embryos under these conditions is shown in Table 2. ND, no detection.
a
Developmental Dynamics
TABLE 2. Survival Rate of Embryos After Microinjectiona Injection
DNA- I-SceI
Developmental stage
volume (nl/egg)
incubation time (min)
n
C (%)
B (%)
N (%)
20 20 4
0 40 40
177 137 114
155(100) 126(100) 107(100)
61(39.4) 65(51.6) 83(77.5)
6(3.9) 10(7.9) 13(12.4)
TB (%)
SL (%)
1(0.6) 5(3.9) 6(5.6)
0(0) 0(0) 2(1.9)b
Injection solution contained the DNA construct (0.1 mg/ml) and I-SceI enzyme (1 U/ ml). Embryos after microinjection were reared at room temperature (22–24 C). n, total number of eggs (one-cell embryos) examined; C, cleavage; B, blastula; N, neurula; TB, tail-bud; SL, swimming larvae. b One of them showed a moderate and uniform expression pattern of EGFP, and the other was mosaic. a
TABLE 3. Influence of Rearing Temperature on the Survival of Poked Embryos
Rearing temperature ( C) a
22–24 18 14
Developmental stage n
C (%)
B (%)
N (%)
TB (%)
SL (%)
48 105 33
34(100) 76(100) 33(100)
20(58.8) 76(100) 29(87.9)
10(29.4) 68(89.5) 27(81.8)
10(29.4) 68(89.5) 27(81.8)
10(29.4) 42(55.3) 27(81.8)
a
Data obtained by tracer injection (circles in Fig. 2D) were arranged for comparison. For abbreviations, see Table 2.
yield optimal results. After stage 35, we could rear animals at room temperature (22–24 C).
Optimal Conditions for Transgenesis With I-SceI in the Newt Subsequently, we tried microinjection again in conjunction with the optimal rearing temperature: we injected 4 nl of the microinjection solution (i.e., 0.4
ng DNA and 0.004 U I-SceI), which was pre-incubated at 37 C for 40 min, into one-cell embryos, and reared them at 14 C until the SL-stage. This rearing temperature obviously improved the survival of embryos after microinjection more than at room temperature, i.e., 22–24 C (Table 4); Survival rate: 47.1% (3.8-fold) at the N-stage, 39.7% (7.1-fold) at the TB-stage, 35.3% (18.6-fold) at the SL-stage. Under these conditions, we successfully obtained transgenic juveniles after metamor-
Fig. 5. Optimization of the time allowing the embryos to recover from mechanical damage by microinjection. One-cell embryos were reared for different periods [1, 3, 9, 24 hr, and 1 (up to stage 35)] at 14 C after a poke with the micropipette tip. Percentage of survivors at the swimming larvae (SL) stage significantly increased as the time for recovery extended. Values are the mean 6 SEM (1 h, N ¼ 3, n ¼ 48; 3 h, N ¼ 3, n ¼ 28; 9 h, N ¼ 3, n ¼ 33; 24 h, N ¼ 3, n ¼ 33; 1, N ¼ 3, n ¼ 33). Statistical differences from the data of 1-hr incubation, examined by the Kruskal-Wallis test followed by Fisher’s LSD test, are indicated by *P
